The aim of plant biotechnology work is the generation of plants with advantageous novel properties, for example for increasing agricultural productivity. The plants' natural defense mechanisms against pathogens are frequently insufficient. Fungal diseases alone result in annual yield losses of many billions of US$. The introduction of foreign genes from plants, animals or microbial sources can increase the defenses. Examples are the protection against feeding damage by insects by expressing Bacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37) or the protection against fungal infection by expressing a bean chitinase (Broglie et al. (1991) Science 254:1194-1197). However, most of the approaches described only offer resistance to a single pathogen or a narrow spectrum of pathogens.
Only a few approaches exist which impart a resistance to a broader spectrum of pathogens to plants. Systemic acquired resistance (SAR)—a defense mechanism in a variety of plant/pathogen interactions —can be conferred by the application of endogenous messenger substances such as jasmonic acid (JA) or salicylic acid (SA) (Ward, et al. (1991) Plant Cell 3:1085-1094; Uknes, et al. (1992) Plant Cell 4(6):645-656). Similar effects can also be achieved by synthetic compounds such as 2,6-dichloroisonicotinic acid (INA) or S-methyl benzo(1,2,3)thiadiazole-7-thiocarboxylate (BTH; Bion200 ) (Friedrich et al. (1996) Plant J 10(1) :61-70; Lawton et al. (1996) Plant J. 10:71-82). The expression of pathogenesis-related (PR) proteins, which are upregulated in the case of SAR, may also cause pathogen resistance in some cases.
In barley, the Mlo locus has been described as a negative regulator of the defense against pathogens. The loss of the Mlo gene causes an increased and, above all, race-unspecific resistance against a large number of mildew species (Büschges R et al. (1997) Cell 88:695-705; Jorgensen J H (1977) Euphytica 26:55-62; Lyngkjaer M F et al. (1995) Plant Pathol 44:786-790). Ml-deficient barley varieties obtained by conventional breeding are already being used in agriculture. Despite intensive cultivation, the resistance has proved to be durable, presumably due to the fact that it is recessive. Mlo-like resistances in other plants, in particular in cereal species, are not described. The Mlo gene and various homologs from other cereal species have been identified and cloned (Büschges R et al. (1997) Cell 88:695-705; WO 98/04586; Schulze-Lefert P, Vogel J (2000) Trends Plant Sci. 5:343-348). Various methods using these genes for obtaining a pathogen resistance are described (WO 98/04586; WO 00/01722; WO 99/47552). The disadvantage is that the Mlo-mediated defense mechanism comprises a spontaneous die-off of leaf cells (Wolter M et al. (1993) Mol Gen Genet 239:122-128). Another disadvantage is that the Mlo-deficient genotypes show hypersensitivity to hemibiotrophic pathogens such as Magnaporte grisea (M. grisea) and Cochliobolus sativus (Bipolaris sorokiniana) (Jarosch B et al. (1999) Mol Plant Microbe Interact 12:508-514; Kumar J et al. (2001) Phytopathology 91:127-133).
The liberation of reactive oxygen species (ROS; for example superoxide (O2−) , hydroxyl radicals and H2O2) is ascribed an important protection function in the reaction on plant pathogens (Wojtaszek P (1997) Biochem J 322:681-692). A variety of ways of how a cell can produce ROS are known. In the macrophages of mammals, it is in particular the enzyme NADPH oxidase, which is able to transfer electrons to molecular oxygen, which must be mentioned. Homologous enzymes have also been identified in plants (Lamb & Dixon (1997) Annu Rev Plant Physiol Plant Mol Biol 48:251).
It has been shown that mutations in the catalytic subunit of NADPH oxidase in Arabidopsis thaliana show a reduced accumulation of reactive oxygen intermediates (ROI). With regard to the hypersensitive reaction (HR), the results were heterogeneous: while infection with the aviralulent and bactrium Pseudomonas syringae showed a reduced HR in a double mutant, the virulent oomycete Peronospora parasitica showed an increased HR. Growth—both of virulent and of avirulent P. syringae strains—was not changed in comparison with wild-type plants, however (Torres M A et al. (2002) Proc Natl Acad Sci USA 99:517-522). Likewise, the inhibition of NADPH oxidase by means of the inhibitor diphenyleneiodonium chloride (DPI)—at physiologically relevant concentrations—had no effect on the development of pathogenic fungi (Hückelhoven R & Kogel K H (1998) Mol Plant Microbe Interact 11:292-300). A cDNA fragment of a phagocytic barley NADPH oxidase (pNAox, homolog of the large subunit gp91phox of a phagocytic NADPH oxidase) is described under the GenBank Acc.-No.: AJ251717).